The transforming growth factor-.beta. (TGF.beta.) polypeptides influence growth, differentiation, and gene expression in many cell types. The first polypeptide of this family that was characterized, TGF.beta.1 has two identical 112 amino acid subunits which are covalently linked. TGF.beta.1 is a highly conserved protein with only a single amino acid difference distinguishing humans from mice. There are two other members of the TGF.beta. gene family that are expressed in mammals. TGF.beta.2 is 71% homologous to TGF.beta.1 (de Martin et al. (1987) EMBO J. 6:3673-3677), whereas TGF.beta.3 is 80% homologous to TGF.beta.1 (Derynck et al. (1988) EMBO J 7:3737-3743). The structural characteristics of TGF.beta.1 as determined by nuclear magnetic resonance (Archer et al. (1993) Biochemistry 32:1164-1171) agree with the crystal structure of TGF.beta.2 (Daopin et al. (1992) Science 257:369-374; Schlunegger and Grutter (1992) Nature 358:430-434) Even though the TGF.beta.'s have similar three dimensional structures, they are by no means physiologically equivalent. There are at least three different extracellular receptors, type I, II and III, involved in transmembrane signaling of TGF.beta. to cells carrying the receptors. (For reviews, see Derynck (1994) TIBS 19:548-553 and Massague (1990) Ann. Rev. Cell Biol. 6:597-641). In order for TGF.beta.2 to effectively interact with the type II TGF.beta. receptor, the type III receptor must also be present (Derynck (1994) TIBS 19:548-553). Vascular endothelial cells lack the type III receptor. Instead endothelial cells express a structurally related protein called endoglin (Cheifetz et al. (1992) J. Biol. Chem. 267:19027-19030), which only binds TGF.beta.1 and TGF.beta.3 with high affinity. Thus, the relative potency of the TGF.beta.'s reflect the type of receptor expressed in a cell and organ system.
In addition to the regulation of the components in the multifactorial signaling pathway, the distribution of the synthesis of TGF.beta. polypeptides also affects physiological function. The distribution of TGF.beta.2 and TGF.beta.3 is more limited (Derynck et al. (1988) EMBO J 7:3737-3743) than TGF.beta.1, e.g., TGF.beta.3 is limited to tissues of mesenchymal origin, whereas TGF.beta.1 is present in both tissues of mesenchymal and epithelial origin.
TGF.beta.1 is a multifunctional cytokine critical for tissue repair. High concentrations of TGF.beta.1 are delivered to the site of injury by platelet granules (Assoian and Sporn (1986) J. Cell Biol. 102:1217-1223). TGF.beta.1 initiates a series of events that promote healing including chemotaxis of cells such as leukocytes, monocytes and fibroblasts, and regulation of growth factors and cytokines involved in angiogenesis, cell division associated with tissue repair and inflammatory responses. TGF.beta.1 also stimulates the synthesis of extracellular matrix components (Roberts et al. (1986) Proc. Natl. Acad. Sci. USA 83:4167-4171; Sporn et al. (1983) Science 219:1329-1330; Massague (1987) Cell 49:437-438) and most importantly for understanding the pathophysiology of TGF.beta.1, TGF.beta.1 autoregulates its own synthesis (Kim et al. (1989) J. Biol. Chem. 264:7041-7045).
A number of diseases have been associated with TGF.beta.1 overproduction. Fibrotic diseases associated with TGF.beta.1 overproduction can be divided into chronic conditions such as fibrosis of the kidney, lung and liver and more acute conditions such as dermal scarring and restenosis. Synthesis and secretion of TGF.beta.1 by tumor cells can also lead to immune suppression such as seen in patients with aggressive brain or breast tumors (Arteaga et al. (1993) J. Clin. Invest. 92:2569-2576). The course of Leishmanial infection in mice is drastically altered by TGF.beta.1 (Barral-Netto et al. (1992) Science 257:545-547). TGF.beta.1 exacerbated the disease, whereas TGF.beta.1 antibodies halted the progression of the disease in genetically susceptible mice. Genetically resistant mice became susceptible to Leishmanial infection upon administration of TGF.beta.1.
The profound effects of TGF.beta.1 on extracellular matrix deposition have been reviewed (Rocco and Ziyadeh (1991) in Contemporary Issues in Nephrology v.23, Hormones, autocoids and the kidney. ed. Jay Stein, Churchill Livingston, New York pp.391-410; Roberts et al. (1988) Rec. Prog. Hormone Res. 44:157-197) and include the stimulation of the synthesis and the inhibition of degradation of extracellular matrix components. Since the structure and filtration properties of the glomerulus are largely determined by the extracellular matrix composition of the mesangium and glomerular membrane, it is not surprising that TGF.beta.1 has profound effects on the kidney. The accumulation of mesangial matrix in proliferative glomerulonephritis (Border et al. (1990) Kidney Int. 37:689-695) and diabetic nephropathy (Mauer et al. (1984) J. Clin. Invest. 74:1143-1155) are clear and dominant pathological features of the diseases. TGF.beta.1 levels are elevated in human diabetic glomerulosclerosis (advanced neuropathy) (Yamamoto et al. (1993) Proc. Natl. Acad. Sci. 90:1814-1818). TGF.beta.1 is an important mediator in the genesis of renal fibrosis in a number of animal models (Phan et al. (1990) Kidney Int. 37:426; Okuda et al. (1990) J. Clin. Invest. 86:453). Suppression of experimentally induced glomerulonephritis in rats has been demonstrated by antiserum against TGF.beta.1 (Border et al. (1990) Nature 346:371) and by an extracellular matrix protein, decorin, which can bind TGF.beta.1 (Border et al. (1992) Nature 360:361-363).
Too much TGF.beta.1 leads to dermal scar-tissue formation. Neutralizing TGF.beta.1 antibodies injected into the margins of healing wounds in rats have been shown to inhibit scarring without interfering with the rate of wound healing or the tensile strength of the wound (Shah et al. (1992) Lancet 339:213-214). At the same time there was reduced angiogenesis, reduced number of macrophages and monocytes in the wound, and a reduced amount of disorganized collagen fiber deposition in the scar tissue.
TGF.beta.1 may be a factor in the progressive thickening of the arterial wall which results from the proliferation of smooth muscle cells and deposition of extracellular matrix in the artery after balloon angioplasty. The diameter of the restenosed artery may be reduced 90% by this thickening, and since most of the reduction in diameter is due to extracellular matrix rather than smooth muscle cell bodies, it may be possible to open these vessels to 50% simply by reducing extensive extracellular matrix deposition. In uninjured pig arteries transfected in vivo with a TGF.beta.1 gene, TGF.beta.1 gene expression was associated with both extracellular matrix synthesis and hyperplasia (Nabel et al. (1993) Proc. Natl. Acad. Sci. USA 90:10759-10763). The TGF.beta.1 induced hyperplasia was not as extensive as that induced with PDGF-BB, but the extracellular matrix was more extensive with TGF.beta.1 transfectants. No extracellular matrix deposition was associated with FGF-1 (a secreted form of FGF) induced hyperplasia in this gene transfer pig model (Nabel (1993) Nature 362:844-846).
There are several types of cancer where TGF.beta.1 produced by the tumor may be deleterious. MATLyLu rat cancer cells (Steiner and Barrack (1992) Mol. Endocrinol. 6:15-25) and MCF-7 human breast cancer cells (Arteaga et al. (1993) Cell Growth and Differ. 4:193-201) became more tumorigenic and metastatic after transfection with a vector expressing the mouse TGF.beta.1. In breast cancer, poor prognosis is associated with elevated TGF.beta. (Dickson et al. (1987) Proc. Natl. Acad. Sci. USA 84:837-841; Kasid et al. (1987) Cancer Res. 47:5733-5738; Daly et al. (1990) J. Cell Biochem. 43:199-211; Barrett-Lee et al. (1990) Br. J Cancer 61:612-617; King et al. (1989) J. Steroid Biochem. 34:133-138; Welch et al. (1990) Proc. Natl. Acad. Sci. USA 87:7678-7682; Walker et al. (1992) Eur. J. Cancer 238:641-644) and induction of TGF.beta.1 by tamoxifen treatment (Butta et al. (1992) Cancer Res. 52:4261-4264) has been associated with failure of tamoxifen treatment for breast cancer (Thompson et al. (1991) Br. J Cancer 63:609-614). Anti TGF.beta.1 antibodies inhibit the growth of MDA-231 human breast cancer cells in athymic mice (Arteaga et al. (1993) J. Clin. Invest. 92:2569-2576), a treatment which is correlated with an increase in spleen natural killer cell activity. CHO cells transfected with latent TGF.beta.1 also showed decreased NK activity and increased tumor growth in nude mice (Wallick et al. (1990) J. Exp. Med. 172:1777-1784). Thus, TGF.beta.1 secreted by breast tumors may cause an endocrine immune suppression.
High plasma concentrations of TGF.beta.1 have been shown to indicate poor prognosis for advanced breast cancer patients (Anscher et al. (1993) N. Engl. J. Med. 328:1592-1598). Patients with high circulating TGF.beta. before high dose chemotherapy and autologous bone marrow transplantation are at high risk for hepatic veno-occlusive disease (15-50% of all patients with a mortality rate up to 50%) and idiopathic interstitial pneumonitis (40-60% of all patients). The implication of these findings is 1) that elevated plasma levels of TGF.beta.1 can be used to identify at risk patients and 2) that reduction of TGF.beta.1 could decrease the morbidity and mortality of these common treatments for breast cancer patients.
A method for the in vitro evolution of nucleic acid molecules with high affinity binding to target molecules has been developed. This method, Systematic Evolution of Ligands by EXponential enrichment, termed SELEX, is described in U.S. Pat. application Ser. No. 07/536,428, filed Jun. 11, 1990, entitled "Systematic Evolution of Ligands by Exponential Enrichment," now abandoned, U.S. patent application Ser. No. 07/714,131, filed Jun. 10, 1991, entitled "Nucleic Acid Ligands," now issued as U.S. Pat. No. 5,475,096, U.S. patent application Ser. No. 07/931,473, filed Aug. 17, 1992, entitled "Methods for Identifying Nucleic Acid Ligands," now U.S. Pat. No. 5,270,163 (see also WO91/19813), each of which is herein specifically incorporated by reference. Each of these applications, collectively referred to herein as the SELEX Patent Applications, describe a fundamentally novel method for making a nucleic acid ligand to any desired target molecule.
The SELEX method involves selection from a mixture of candidate oligonucleotides and step-wise iterations of binding, partitioning and amplification, using the same general selection theme, to achieve virtually any desired criterion of binding affinity and selectivity. Starting from a mixture of nucleic acids, preferably comprising a segment of randomized sequence, the SELEX method includes steps of contacting the mixture with the target under conditions favorable for binding, partitioning unbound nucleic acids from those nucleic acids which have bound to target molecules, dissociating the nucleic acid-target complexes, amplifying the nucleic acids dissociated from the nucleic acid-target complexes to yield a ligand-enriched mixture of nucleic acids, then reiterating the steps of binding, partitioning, dissociating and amplifying through as many cycles as desired to yield high affinity nucleic acid ligands to the target molecule.
The basic SELEX method may be modified to achieve specific objectives. For example, U.S. patent application Ser. No. 07/960,093, filed Oct. 14, 1992, entitled "Method for Selecting Nucleic Acids on the Basis of Structure," now abandoned, describes the use of SELEX in conjunction with gel electrophoresis to select nucleic acid molecules with specific structural characteristics, such as bent DNA. (See U.S. Pat. No. 5,707,796). U.S. patent application Ser. No. 08/123,935, filed Sep. 17, 1993, entitled "Photoselection of Nucleic Acid Ligands," now abandoned, describes a SELEX based method for selecting nucleic acid ligands containing photoreactive groups capable of binding and/or photocrosslinking to and/or photoinactivating a target molecule. U.S. patent application Ser. No. 08/134,028, filed Oct. 7, 1993, entitled "High-Affinity Nucleic Acid Ligands That Discriminate Between Theophylline and Caffeine," abandoned in favor of U.S. patent application Ser. No. 08/443,957, now U.S. Pat. No. 5,580,737,describes a method for identifying highly specific nucleic acid ligands able to discriminate between closely related molecules, termed "Counter-SELEX." U.S. patent application Ser. No. 08/143,564, filed Oct. 25, 1993, entitled "Systematic Evolution of Ligands by EXponential Enrichment: Solution SELEX," abandoned in favor of U.S. patent application Ser. No. 08/461,061, now U.S. Pat. No. 5,567,588) and U.S. patent application Ser. No. 08/792,075, filed Jan. 31, 1997, entitled "Flow Cell SELEX," describe SELEX-based methods which achieve highly efficient partitioning between oligonucleotides having high and low affinity for a Target molecule. U.S. patent application Ser. No. 07/964,624, filed Oct. 21, 1992, entitled "Nucleic Acid Ligands to HIV-RT and HIV-1 Rev," now U.S. Pat. No. 5,496,938, describes methods for obtaining improved Nucleic Acid Ligands after the SELEX process has been performed. U.S. patent application Ser. No. 08/400,440, filed Mar. 8, 1995, entitled "Systematic Evolution of Ligands by EXponential Enrichment: Chemi-SELEX," describes methods for covalently linking a ligand to its target.
The SELEX method encompasses the identification of high-affinity nucleic acid ligands containing modified nucleotides conferring improved characteristics on the ligand, such as improved in vivo stability or delivery. Examples of such modifications include chemical substitutions at the ribose and/or phosphate and/or base positions. Specific SELEX-identified nucleic acid ligands containing modified nucleotides are described in U.S. patent application Ser. No. 08/117,991, filed Sep. 8, 1993, entitled "High Affinity Nucleic Acid Ligands Containing Modified Nucleotides," abandoned in favor of U.S. patent application Ser. No. 08/430,709, now U.S. Pat. No. 5,660,985, that describes oligonucleotides containing nucleotide derivatives chemically modified at the 5- and 2'-positions of pyrimidines, as well as specific RNA ligands to thrombin containing 2'-amino modifications. U.S. patent application Ser. No. 08/134,028, supra, describes highly specific nucleic acid ligands containing one or more nucleotides modified with 2'-amino (2'-NH.sub.2), 2'-fluoro (2'-F), and/or 2'-O-methyl (2'-OMe). U.S. patent application Ser. No. 08/264,029, filed Jun. 22, 1994, entitled "Novel Method of Preparation of Known and Novel 2' Modified Nucleosides by Intramolecular Nucleophilic Displacement," describes oligonucleotides containing various 2'-modified pyrimidines. International patent application PCT/US98/00589, filed Jan. 7, 1998, entitled "Bioconjugation of Oligonucleotides," describes a method for identifying bioconjugates to a target comprising nucleic acid ligands derivatized with a molecular entity exclusively at the 5'-position of the nucleic acid ligands.
The SELEX method encompasses combining selected oligonucleotides with other selected oligonucleotides and non-oligonucleotide functional units as described in U.S. patent application Ser. No. 08/284,063, filed Aug. 2, 1994, entitled "Systematic Evolution of Ligands by Exponential Enrichment: Chimeric SELEX," now U.S. Pat. No. 5,637,459 and U.S. patent application Ser. No. 08/234,997, filed Apr. 28, 1994, entitled "Systematic Evolution of Ligands by Exponential Enrichment: Blended SELEX," now U.S. Pat. No. 5,683,867, respectively. These applications allow the combination of the broad array of shapes and other properties, and the efficient amplification and replication properties of oligonucleotides with the desirable properties of other molecules. The full text of the above described patent applications, including but not limited to, all definitions and descriptions of the SELEX process, are specifically incorporated herein by reference in their entirety.